Although radiation therapy represents a critical modality in the treatment of numerous malignancies, it can be associated with substantial long-term morbidity. There is growing interest in personalizing treatment strategies to reduce patient toxicity while maintaining or improving oncologic outcomes. HPV-associated malignancies of the oropharynx, cervix and anus represent an ideal group of diseases for this approach. For these sites, radiation therapy is the preferred treatment modality for primary disease but the anatomic locations predispose patients to significant morbidity. Molecular biomarkers of treatment response and/or outcome are being increasingly applied to clinical practice. However, such signatures fail to include a consideration of treatment-associated toxicity. Ideally, the selection of personalized therapy should not only incorporate an estimate of treatment success but also of potential morbidity. Molecular changes such as DNA methylation have been shown to play an important role in the progression of HPV-associated malignancies. Furthermore, such tumors appear to share a broad number of discrete methylation events and we have postulated that certain common alterations may in fact, contribute to the regulation of sensitivity to radiation treatment and its associated toxicities. Indeed, in our preliminary studies of ~120 anal cancer specimens, using genome-wide methylation array profiling, we have demonstrated that there are differentially methylated DNA sites that distinguish between: 1) Responders and non-responders to standard chemo-radiation therapy and 2) Patients suffering moderate to severe morbidity and those with little- to-no long term radiation toxicities. We hypothesize that methylation signatures separately predictive of radiation response and toxicity can be identified and are commonly applicable among HPV-associated SCCs. In Aims 1 and 2, we will develop robust methylation signatures predictive of radiation response (Aim 1) and long-term toxicity (Aim 2) among 260 anal cancer cases from the Radiation Therapy Oncology Group (RTOG) 98-11 national anal cancer clinical trial using a training/test set approach. In Aim 3, we will determine the applicability of the two anal cancer-derived signatures to HPV-associated cervical (n=90) and oropharyngeal cancers (n=100). We envision that the combined application of these signatures will ultimately represent a unique and powerful tool that would allow the classification of patients into discrete groups stratified by low/high radio-sensitivity and low/high risk of toxiity and could form the basis for personalized treatment strategies. By evaluating the interplay between the epigenome, radiation response and treatment morbidity in HPV- associated cancers, we will develop instruments that can ultimately be applied towards personalized dose modification and toxicity reduction with the ultimate goal of improving patient outcomes and quality of life.